Genetics and Mechanism of Pheochromocytoma–Paraganglioma Syndromes Characterized by Germline SDHB and SDHD Mutations

Home , ISCU, SDHB, SDHD, Succinate dehydrogenase

Role of SDH in B E Baysal and E R Maher 22:4 T71–T82 Thematic Review pheochromocytomas/ paragangliomas

15 YEARS OF PARAGANGLIOMA Genetics and mechanism of pheochromocytoma–paraganglioma syndromes characterized by germline SDHB and SDHD mutations

Bora E Baysal and Eamonn R Maher1 Correspondence should be addressed Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA to B E Baysal 1Department of Medical Genetics, Cambridge NIHR Biomedical Research Centre, Email University of Cambridge, Cambridge CB2 0QQ, UK [email protected]

Abstract

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine neoplasms that Key Words derive from small paraganglionic tissues which are located from skull base to the pelvic ﬂoor. " pheochromocytoma Genetic predisposition plays an important role in development of PPGLs. Since the discovery " neuroendocrine tumors of ﬁrst mutations in the succinate dehydrogenase D (SDHD) gene, which encodes the " neoplasia smallest subunit of mitochondrial complex II (SDH), genetic studies have revealed a major " molecular genetics role for mutations in SDH subunit genes, primarily in SDHB and SDHD, in predisposition to " molecular biology

Endocrine-Related Cancer both familial and non-familial PPGLs. SDH-mutated PPGLs show robust expression of hypoxia induced genes, and genomic and histone hypermethylation. These effects occur in part through succinate-mediated inhibition of a-ketoglutarate-dependent dioxygenases. However, details of mechanisms by which SDH mutations activate hypoxic pathways and trigger subsequent neoplastic transformation remain poorly understood. Here, we present

a brief review of the genetic and mechanistic aspects of SDH-mutated PPGLs. Endocrine-Related Cancer (2015) 22, T71–T82

Introduction

Paragangliomas are neuroendocrine neoplasms that may (Tischler 2008). Thus they are functionally and histolo- arise from parasympathetic or sympathetic paraganglia. In gically similar to pheochromocytomas (in older literature general, those arising from parasympathetic paraganglia they were often described as extra-adrenal pheochromo- are non-secretory, associated with head and neck para- cytomas). Occasionally HNPGL can also secrete catechol- ganglia and are usually referred to as head and neck amines (Erickson et al. 2001). paragangliomas (HNPGL), or more specifically ‘carotid Familial HNPGL was first described more than 80 years paraganglioma’, ‘jugulotympanic paraganglioma’, etc., ago (Chase 1933), and 25 years ago evidence of autosomal rather than by older terms such as ‘chemodectoma’ or dominant inheritance with parent of origin effects ‘glomus jugulare’. Paragangliomas arising from sym- (tumors were only manifest after paternal transmission, pathetic nervous system paraganglia usually arise in the Fig. 1) was reported (van der Mey et al. 1989). Ten years abdomen and thorax, and secrete catecholamines later, Baysal et al. (2000) reported the seminal finding that

http://erc.endocrinology-journals.org q 2015 Society for Endocrinology This paper is part of a thematic review section on 15th Anniversary of Paraganglioma DOI: 10.1530/ERC-15-0226 Printed in Great Britain and Pheochromocytoma. The Guest Editors for this section were Wouter de Herder Published by Bioscientiﬁca Ltd. and Hartmut Neumann. Downloaded from Bioscientifica.com at 09/24/2021 06:18:29AM via free access Role of SDH in Thematic Review B E Baysal and E R Maher 22:4 T72 pheochromocytomas/ paragangliomas

juvenile encephalopathy (Bourgeron et al. 1995), were demonstrated to be a rare cause of (dominantly inherited) predisposition to PPGL (though penetrance appears to be very low) (Burnichon et al.2010). Though germline mutations in SDHC have been shown to be associated on rare occasions with PPGL (Mannelli et al.2007), these are much less frequent than SDHB and SDHD mutations. Genetic heterogeneity of PPGLs is further highlighted by identiﬁcation of germline mutations in the VHL, RET, NF1, TMEM127,andMAX genes (Dahia 2014). Here we review our current knowledge of SDHB-and

Affected mutation carrier SDHD-related disorders.

Unaffected non-mutation carrier

Unaffected mutation carrier Germline SDHB and SDHD mutations Mutation spectrum Figure 1 A comprehensive database of germline SDHB and SDHD Sample family tree illustrating how disease can be hidden within families due to maternal imprinting of the SDHD gene. The susceptibility gene is mutations is maintained at http://chromium.liacs.nl/ carried and transmitted by females but only manifests as disease when LOVD2/SDH/home.php (Bayley et al. 2005). A wide variety inherited from a father. of intragenic mutations have been described and, more recently, single or multiple exon deletions (and, occasion- familial HNPGL was associated with germline mutations ally, intragenic duplications; McWhinney et al. 2004, in succinate dehydrogenase D (SDHD; PGL1 locus) and Cascon et al. 2008, Neumann et al. 2009). A number of subsequently, SDHD mutations were also demonstrated to frequent SDHB and SDHD mutations were observed and be associated with sporadic and familial pheochromocy- these may result from a high mutation rate or to founder toma (Gimm et al. 2000, Astuti et al. 2001a). SDHD encodes effects. Thus the relative frequency of some mutations can the D subunit of the SDH heteterotetrameric enzyme that, vary with geographical location. In the Netherlands, two Endocrine-Related Cancer together with SDHC, anchors the SDH complex to the inner major SDHD founder mutations have been identified mitochondrial inner membrane. SDH has critical roles in (c.274GOT (p.Asp92Tyr) and c.416TOC (p.Leu139Pro)), the Krebs cycle and respiratory chain electron transport (as and these account for O90% of SDHD mutation carriers part of mitochondrial complex II, Fig. 2). The SDHB gene product, containing three iron–sulfur clusters, is part of the hydrophilic catalytic domain and binds to the SDHA gene SDHA product that contains a covalently attached flavin adenine SDHAF2 dinucleotide (FAD) co-factor and the substrate binding site. SDHB SDHB and SDHD gene products bind to each other and attach the complex II holoenzyme to the mitochondrial inner membrane. Soon after the associations of SDHD mutations with human disease, germline SDHC (PGL3) SDHD mutations were reported to cause familial HNPGL (inher- SDHC ited as an autosomal dominant trait without parent- of-origin effects) (Niemann & Muller 2000) and germline SDHB (PGL4) mutations were found to cause inherited Figure 2 susceptibility to HNPGL and pheochromocytomas and Schematic structure of SDH subunits is shown. SDH is comprised of four structural subunits encoded by SDHA, SDHB, SDHC,andSDHD. The SDHC paragangliomas (PPGL) (Astuti et al. 2001b). Subsequently and SDHD gene products are hydrophobic, sandwich a heme moiety and germline mutations in the SDH-associated protein span the inner mitochondrial membrane. The SDHA and SDHB gene SDHAF2 were found to be a rare cause of HNPGL products are cytosolic and contain a covalently bound FAD and three iron–sulfur clusters respectively. Germ line mutations in SDHAF2 (SDH5), (Hao et al. 2009, Bayley et al. 2010) and SDHA mutations, which encode a non-structural assembly protein critical for flavination of initially reported in the context of an autosomal recessive the SDHA gene product, also predispose to PPGL.

http://erc.endocrinology-journals.org q 2015 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/ERC-15-0226 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 06:18:29AM via free access Role of SDH in Thematic Review B E Baysal and E R Maher 22:4 T73 pheochromocytomas/ paragangliomas

(van Hulsteijn et al. 2012). A SDHD c.33C/A (p.Cys11X) preservation of IGF2 expression from the paternal allele founder mutation has been reported in central Europe (Hensen et al.2004, Margetts et al.2005). In contrast, it can (Poland; Peczkowska et al. 2008). The common SDHD be hypothesized that, in individuals harboring a maternally c.242COT (p.Pro81Leu) mutation has been reported as inherited SDHD mutation, loss of the paternally-derived both a recurrent and a founder mutation (Baysal et al. chromosome 11 would, whilst biallelically inactivating 2002). In the Dutch population, SDHB founder mutations SDHD,resultinlossofIGF2 expression and retention of arelesscommon.Themostfrequent(asplicesite CDKN1C expression. Such a combination is not usually mutation c.423C1G) intragenic mutation was about 15 sufficient to drive tumorigenesis. In support of this timeslesscommonthantheSDHD c.274GOT hypothesis is the observation that in one case of para- (p.Asp92Tyr) founder mutation, and a founder SDHB ganglioma after maternal transmission of a SDHD mutation, exon 3 deletion has also been reported (Bayley et al. there was loss of the paternal SDHD allele and loss of the 2009a,b, Hensen et al. 2012). In Spain, SDHB founder maternal 11p15.5 imprinted region (Yeap et al.2011). mutations (exon 1 deletion and SDHB c.166_170delCCT- An alternative model to explain the parent-of-origin CA) have also been reported (Cascon et al. 2008, 2009). effects in transmission of SDHD-related paragangliomas suggests regulation of SDHD gene expression by a long- range epigenetic mechanism (Baysal et al. 2011). This Penetrance and genotype–phenotype correlations model proposes that an imprinted small CpG island A major difference between the clinical presentation of associated with a long intergenic non-coding RNA at the germline SDHB and SDHD mutations is the parent-of-origin boundary of gene-rich SDHD domain regulates availability effect with the latter. Apart from a few exceptional cases of a hypothetical distal enhancer to the SDHD promoter. in which clinical disease has developed after maternal Particularly for germline SDHB mutations, the transmission of a SDHD mutation (Yeap et al.2011), the risk increased use of presymptomatic genetic testing in of clinical disease after a maternal transmission appears extended families has resulted in recognition that the to be extremely remote. Notably, the paraganglioma penetrance of SDHB mutations is lower than initially phenotype in such cases appears mild or atypical (e.g. no thought. Thus initial estimates of the penetrance of multi-focal tumors) indicating functional inequality of germline SDHB mutations were in excess of 70% but the two parental alleles in tumor pathogenesis (reviewed have progressively fallen to 25–40% (Benn et al. 2006, Solis in Baysal (2013)). Though to date unequivocal evidence of et al. 2009, Hes et al. 2010, Ricketts et al. 2010, Schiavi et al. Endocrine-Related Cancer genomic imprinting at the SDHD locus has not been found, 2010). The relatively low penetrance of SDHB mutations is paraganglia-specific partial (quantitative) imprinting of consistent with the observation of a low de novo mutation SDHD cannot be excluded. Differential methylation of a rate, frequent founder mutations and the relatively high minor CpG island upstream of a long non-coding RNA number of mutations detected in apparently isolated cases located at the telomeric boundary of gene-rich SDHD (Baysal et al. 2002, Neumann et al. 2002, Cascon et al. domain was proposed to regulate long-range enhancer- 2009, Jafri et al. 2013). However, the low penetrance can promoter interactions (Baysal et al.2011). make the interpretation of likely pathogenicity for a novel Homozygous SDHD mutations have been associated sequence variant detected in individuals with a potentially with recessively inherited encephalomyopathy and mito- SDH-related neoplasm complex and also raises, as yet chondrial complex II deficiency (Jackson et al. 2014). unresolved questions, as to the type and intensity of Tumorigenesis in SDH-mutated neoplasia appears to follow tumor surveillance in asymptomatic gene carriers. a ‘two hit’ (retinoblastoma-like) model and it has been Though SDHB and SDHD encode components of the proposed that the parent-of-origin effects may reflect the same protein complex, there are some differences in the tendency for the ‘second hit’ causing inactivation of the WT relative propensities for developing different tumor types. allele in SDHD-related tumorigenesis to be loss of the whole Thus SDHD mutations are generally associated with a chromosome 11. The imprinted gene cluster at 11p15.5 higher risk of HNPGL than non-HNPGL. For SDHB contains the maternally expressed growth suppressor mutations, extra-adrenal and non-HNPGL is more often CDKN2B and the paternally expressed IGF2 growth factor the presenting feature than HNPGL or pheochromocy- (Lim & Maher 2010). In cases of a paternally inherited toma, and there is a significantly higher risk of malignant germline SDHD mutation, loss of the maternally-derived paraganglioma and poor prognosis (w25% lifetime risk; chromosome 11 would, in a single event, result in biallelic Gimenez-Roqueplo et al. 2003, Amar et al. 2007, Ricketts SDHD inactivation and loss of CDKN1C expression but et al. 2010). Despite the heterogeneity of SDHB mutations,

there are no clear genotype–phenotype correlations but targeting higher risk subgroups (Erlic et al. 2009). The for SDHD, though it has been suggested that the common application of such testing protocols was assessed in an p.Pro81Leu mutation is associated with a very low risk of audit of a referral-based testing series of SDHB, SDHD, and PPGL (in contrast to truncating SDHD mutations for VHL (Jafri et al. 2013), and it was demonstrated that which the risk is closer to that seen with germline SDHB though widening the testing criteria for testing sporadic mutations; Ricketts et al. 2010). pheochromocytoma cases (e.g. from only those aged !45 A small number of additional tumor types have been years at diagnosis to those aged !60 years) increased the reported in individuals with germline SDHB and SDHD numbers of mutation carriers tested but the cost of mutations. Gastrointestinal tumors (GIST) are the best detecting each mutation carrier increased. A comp- defined association. Carney–Stratakis syndrome is charac- lementary approach is to undertake immunohistochem- terized by the association of GIST with paraganglioma and, in ical analysis for SDHB protein expression in the tumors of most cases is caused by mutations in SDHX genes patients who fall outside the selection criteria. Though (McWhinney et al. 2007, Janeway et al.2011). Germline loss of SDHB expression is a sensitive and specific indicator SDHX mutation may also be detected in patients with familial of germline SDHX mutations (van Nederveen et al. 2009), or sporadic nonsyndromic WT GIST (Janeway et al.2011). tumor material may not always be available to evaluate Renal tumors have been reported, predominantly protein loss. However, as additional inherited HNPGL/ with SDHB mutations, but also with SDHD/SDHC PPGL genes have been identified, there has been increas- mutations and may be the presenting feature in patients ing interest in the application of next generation without a personal or family history of HNPGL/PPGL sequencing strategies to allow comprehensive and less (Vanharanta et al. 2004, Ricketts et al. 2008, 2010). A expensive genetic analysis. Thus specific targeted rese- variety of histopathologies may occur (e.g. conventional quencing panels and exome analysis strategies have been (clear cell), papillary and oncocytoma) and the lifetime described (Rattenberry et al. 2013, McInerney-Leo et al. risk of renal tumors in SDHB mutation carriers has been 2014). As the cost of genetic analysis falls, it seems likely estimated to be up to 15% (Ricketts et al. 2010). that there will be a move towards more extensive analysis. Recently, a clinical association between pituitary adenoma and PPGL has been recognized. Molecular Pathogenesis of SDH-mutated PPGLs genetic studies have shown that this association may be caused by a variety of germline mutations in known PPGL Pathogenesis of PPGLs caused by SDH mutations remains Endocrine-Related Cancer predisposition genes (e.g. SDHB, SDHD, SDHC, VHL, and poorly understood. SDH catalyzes the oxidation of MEN1), or may be sporadic, but the most frequently succinate to fumarate in the Krebs cycle and functions as implicated genes are SDH-subunit genes (Xekouki et al. mitochondrial complex II by transferring the extracted 2011, Papathomas et al. 2013, Deńes et al. 2015). electrons to ubiquinone in the electron transport chain. Loss of SDH activity leads to increased succinate and reactive oxygen species (ROS). Thus, succinate and ROS are Application of SDHB and SDHD mutation testing in considered as the signaling molecules that ultimately clinical practice trigger tumor formation upon SDH mutations. Since The recognition that a substantial proportion (approxi- discovery of the first mutations in familial PPGLs in mately one-quarter of apparently sporadic cases 2000–2001 (Baysal et al. 2000, Niemann & Muller 2000, (Neumann et al. 2002)) might harbor a germline mutation Astuti et al. 2001b), alternative models for tumor in SDHB, SDHD, VHL,orRET led to suggestions that all development have been advanced using different obser- PPGL patients might be offered genetic testing. However, vations and experimental models that studied the such an approach, particularly prior to the application of consequences of SDH genetic loss. These models can be next generation sequencing techniques, was expensive broadly classified as constitutive hypoxic drive, inhibition and did not take into account the how clinical indicators of developmental neuronal culling and histone/genome can be used. In particular, family history of HNPGL/PPGL, hypermethylation (Fig. 3). multiple tumors, extra-adrenal location, or early age at diagnosis (mean age at diagnosis in SDHB/SDHD-related Constitutive hypoxic drive tumors is w10 years earlier than in sporadic cases) can be used to stratify the likelihood of a germline mutation The most common phenotypic manifestation of germline being detected and so increase cost-effectiveness by SDH mutations is the development of PPGL tumors

HIF

PhDs HIF-dependent

HIF OH Hypoxia CoQ signaling Citric acid Succinate – FAD cycle 2 e Succinate/ROS Fumarate SCNR1 HIF-independent ???

Inhibition of PhD3 apoptosis Non-hypoxia Histone signaling HMTs/TETs and DNA methylation

Figure 3 Overview of the proposed mechanisms of SDH-mutated paragangliomas. culture and animal models. Succinate may separately stimulate certain Mutations in SDH subunits (mainly in SDHB and SDHD) result in loss of biological pathways regulated by succinate receptors (e.g. SCNR1). complex II activity and drives paraganglioma formation through accumu- Alternatively, succinate-mediated inhibition of certain a-KG-dependent lation of ROS or succinate. Evidence favors constitutive hypoxia signaling as enzymes such as PhD3, histone methyl transferases (HMTs), or TETs may lead the initiating mechanism of paraganglioma formation. Role of HIF1a/HIF2a to inhibition of neural apoptosis, histone, and DNA hypermethylation. in mediating this hypoxic signaling remains to be conﬁrmed in relevant cell

(Neumann et al. 2004, Dahia 2014). Gastrointestinal Gene expression proﬁles of SDH-mutated PPGLs stromal tumors (Janeway et al. 2011, Pantaleo et al. 2011) Recent genome-wide expression proﬁling studies show and renal carcinoma (Neumann et al. 2004, Ricketts et al. strong induction of hypoxia and angiogenesis pathways 2008) also develop in a small minority of subjects who in SDH- and VHL-related PPGL (Dahia et al. 2005, carry germ line SDH mutations. HNPGL, especially the Lopez-Jimenez et al. 2010, Shankavaram et al. 2013). Endocrine-Related Cancer carotid body (CB) paraganglioma, are characteristically SDH and VHL mutations induce both protein encoding associated with germ line mutations in structural subunit mRNAs and miRNAs (miR-210; Tsang et al. 2014) that are genes SDHD, SDHC, SDHB, and in regulatory subunit implicated in cellular adaptation to hypoxia. Although genes SDHAF2 (Boedeker et al. 2014). The CB is an acute certain differences in the induced genes were observed, the oxygen-sensing organ thatrespondstohypoxiaby broad overlap amongst the hypoxia induced genes increasing heart and ventilation rate (Lopez-Barneo et al. between SDH- and VHL-related paragangliomas strongly 2008). It has been recognized that the incidence of CB suggest that pathogenesis of SDH tumors involves paragangliomas increase among high altitude dwellers constitutive hypoxic stimulation. (Saldana et al. 1973) and those with chronic cyanotic heart The VHL gene product (pVHL) is a component of the diseases (Lack 1978, Opotowsky et al.2015). These protein complex that possesses ubiquitin ligase activity observations suggested early on that the SDH mutations which mediates the proteosomal degradation of disrupt oxygen sensing of the CB by causing an inability to hypoxia-inducible factors (HIFs) under normoxia (Gos- register presence of normal oxygen levels (Baysal et al. sage et al. 2015). HIFs (HIF1, HIF2, and HIF3) are 2000). The paraganglioma tumor formation may thus transcription factors that mediate cellular adaptation to follow chronic hypoxic stimulation of the CB oxygen- hypoxia (Semenza 2012). HIFa subunits are hydroxylated sensing (chief) cells, either by environmental hypoxia or by prolyl or asparaginyl hydroxylase enzymes (PhD1, by SDH mutations that inhibit oxygen sensing. The PhD2, PhD3, and FIH) in normoxia and subsequently hypothesis that chronic (pseudo)hypoxic stimulation degraded by ubiquitination (Kaelin & Ratcliffe 2008). may lead to hereditary paragangliomas is also supported Hypoxia inhibits the hydroxylase enzymes and leads to by evidence that links increased altitudes to increased stabilization of HIFas. It is thought that mutations in severity of SDH-mutated paraganglioma tumors (Astrom VHL lead to tumor formation through constitutive et al. 2003, Cerecer-Gil et al. 2010). stabilization of HIFs.

Role of HIFs in SDH-mutated PPGLs It thus appears that gene expression studies do not provide an unequivocal evidence for involvement of HIFs Broad transcriptional overlap between SDH and VHL- in pathogenesis of SDH-mutated paragangliomas. Whether related PPGL, and constitutive activation of the HIFs in HIFs play a role in SDH–paraganglioma formation, however, VHL suggested that HIFs may also mediate tumor remains an important question which may not be con- formation in SDH-mutated paragangliomas. HIF1a clusively answered by gene/protein expression studies alone. and/or HIF2a were detected by immunohistochemistry It is important to note that hereditary renal tumors that in both SDH-mutated and sporadic HNPGL (Pollard et al. result from fumarate hydratase (FH) germline mutations 2006, Favier et al. 2009, Merlo et al. 2012). In vitro studies were initially shown to stabilize HIF1a (Pollard et al.2005). using cell lines showed that siRNA-mediated knockdown Accordingly, the knockdown of FH in cell lines led to robust of SDH subunits led to stabilization of HIF1a (Selak et al. stabilization of HIF1a through fumarate mediated inhibition 2005, Cervera et al. 2008, Guzy et al. 2008). These studies of the PhD enzymes (Isaacs et al. 2005). These findings are linked increased succinate or ROS levels to the stabil- similar to the observations previously described for ization of HIFas. Despite these in vitro studies, discordant SDH-mutated pathology: i) HIFas are variably detected in results are obtained from gene expression analyses on the SDH-mutated paragangliomas by gene expression and role of HIFs in SDH-mutated PGL tumors. Although immunohistochemical studies and ii) succinate inhibition significant overlap in gene expression patterns of SDH- of PhD enzymes stabilizes HIF1a upon SDH knockdown in VHL and -related PGL tumors was observed including certain cell lines. Although such observations initially a increased HIF2 , VEGF and reduced electron transport suggested a role for HIF1 in tumor predisposition caused by chain genes by transcriptome-wide studies, HIF target- FH mutations, deletion of the Hif1a gene in the Fh1-deficient gene overexpression and increased glycolysis, as assessed mice, which develops renal cysts, worsened the cystic by such genes as hexokinase II (HK2), lactate dehydro- phenotype (Adam et al. 2011). Thus, Hif1 may not mediate genase, MIR210, PHD3 (EGLN3), ENO1, and SLC2A1 were the cystic renal pathology in the Fh1-mice. These results primarily observed in VHL-mutated paragangliomas imply that HIF stabilization observed in SDH-mutated (Favier et al. 2009, Lopez-Jimenez et al. 2010). In fact, tumors may not necessarily indicate its causative role in overexpression of 67 HIF target genes was sufficient to tumor pathogenesis. Ultimately, animal or cell culture distinguish VHL- from SDHB-mutated pheochromocyto- models that link inactivation of SDH to PPGL development mas (Lopez-Jimenez et al. 2010). Conversely, HNPGLs that or to a hypoxia-related physiological response will be Endocrine-Related Cancer overexpress HIF1a and its target genes were found to have required to evaluate the role of HIFs in SDH-mutated tumor WT SDH sequences and a subset of them was indeed found pathogenesis or SDH-regulated hypoxia response. to carry somatic VHL mutations (Merlo et al. 2012, 2013). Heterozygous inactivation of Sdhb or Sdhd genes in mice In addition, recent sequence and functional studies does not lead to tumor development, in particular while identified somatic mutations in EPAS1 which encodes the homozygous inactivation is embryonic lethal (Bayley et al. HIF2a subunit in sporadic (mostly non-head and neck) 2009a,b, Piruat & Millań-Ucle´s 2014). Sdhd conditional paragangliomas, a subset of which was accompanied by constitutional or paraganglia-confined homozygous polycythemia (Zhuang et al. 2012, Comino-Mendez et al. deletions also show no evidence of tumor development 2013, Toledo et al. 2013). Missense mutations in VHL, (Diaz-Castro et al.2012). These findings highlight species- EPAS1 (HIF2A), and PHD2 are associated with erythrocy- specific differences in tumor susceptibility between human tosis. For example, endemic Chuvash polycythemia is and mouse that follows the inactivation of mitochondrial caused by certain VHL missense germ line mutations (Lee complex II subunits. While gene knockout studies in mice & Percy 2011). In contrast, SDHX mutations have yet to be did not recapitulate the paraganglioma tumor phenotype, associated with erythrocytosis. Gene expression profiling they provide some information on activation of hypoxia- shows that EPAS1-mutated paragangliomas cluster with related pathways. Heterozygous Sdhd deletion increases SDH and VHL-related paragangliomas, and strengthens sensitivity of the CB chief cells to hypoxia (Piruat et al. the role of constitutive hypoxic signaling in pathogenesis 2004),whichisconsistentwiththehypothesisthat of SDH-mutated paragangliomas (Comino-Mendez et al. inactivation of Sdh hampers the ability of CB chief cells to 2013). However, the association with erythrocytosis register normal oxygen levels and triggers normoxic suggests that pathogenesis of EPAS1-mutated paraganglio- activation of hypoxia sensing pathways. Gene expression K K mas is more closely associated with the VHL-mutated analyses of Sdhd / tissues show mixed evidence of K K paragangliomas rather than with the SDH-mutated ones. Hif activation. While Sdhd / MEFs showed Hif1a

stabilization, two other tissues did not show any evidence mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 of hypoxic pathway activation (Millań-Ucle´s et al.2014). in low-grade gliomas, secondary glioblastomas, various In summary, mimicry of the hypoxia-associated CB sarcomas, and acute myeloid leukemia cause increased paragangliomas and gene expression profiling studies fumarate and D-2-hydroxyglutarate, respectively, and lead provide strong evidence of constitutive hypoxic pathway to DNA and histone methylation by inhibiting activation in pathogenesis SDH-mutated paraganglioma a-KG-dependent dioxygenases (Morin et al. 2014). tumors. However, determining the role of HIFs in mediating a-KG-dependent dioxygenases comprise a large family of this hypoxia-driven pathogenesis requires further studies. enzymes that perform diverse and important biological functions including protein modification, repair of alky- lated DNA/RNA, and lipid metabolism. It is notable that Inhibition of a-ketoglutarate dependent dioxygenases there is no major overlap amongst the tumor spectra The PhD enzymes are members of a large family of Fe(II)/ associated with SDH, FH, and IDH1/2 mutations, although a-ketoglutarate (KG)-dependent dioxygenases (Hausinger rare familial paraganglioma cases carrying FH germ line 2004). Inhibition of PhDs by succinate on genetic and mutations have been recently described (Letouze´ et al. pharmacologic inhibition of SDH raised the possibility that 2013, Clark et al. 2014). Thus, succinate, fumarate, and other dioxygenases may also contribute to paraganglioma D-2-hydroxyglutare, the oncometabolites generated by development. Succinate accumulation by SDH inhibition SDH, FH, and IDH1/2 mutations, respectively, may inhibit has been shown to inhibit jumonji-domain histone not only PHD, TET, and histone demethylating enzymes demethylases (JmjC), leading to histone H3 hypermethy- but also other a-KG-dependent dioxygenases that provide lation (Smith et al.2007). Succinate is also shown to inhibit an advantage for cancer cell survival. other a-KG-dependent dioxygenases, including collagen Whether succinate receptors (SUCNR) mediate signal- prolyl-4-hydroxylases and the ten-eleven translocation ing in pathogenesis of SDH-mutated paragangliomas (TET) family of 5-methlycytosine (5mC) hydroxylases, remains an underexplored area. Succinate is a ligand for which leads to hypermethylation of CpG islands (Xiao GPR91 (also known as SUCNR1), a G-protein coupled et al. 2012). Both histone and DNA hypermethylation have receptor (He et al. 2004). SUCNR1 is expressed in kidney, the potential to alter gene expression levels. Examination of liver, spleen and white adipose tissue (Ariza et al. 2012). SDH-mutated paragangliomas showed downregulation of SUCNR1 stimulates angiogenesis in retina. Extracellular gene expression for 191 genes that acquired promoter succinate activates the SUCNR and may increase the VEGF Endocrine-Related Cancer methylation as a result of inhibition of the TET family of levels through HIF1a-independent mechanisms (Sapieha 5mC hydroxylases (Letouze´ et al.2013). Certain methylated et al. 2008). SUCNR1 may mediate certain effects of hypoxia genes including PNMT and KRT19 were linked to neuro- which increases the succinate levels. These results suggest endocrine differentiation and epithelial-to-mesenchymal that succinate can act as a physiological signaling molecule differentiation, respectively, raising the possibility that and raise the possibility that some aspects of paraganglio- succinate-mediated inhibition of TET family 5mC hydro- mas, such as increased vascularity may be mediated by xylases may play a role in SDH-mutated paraganglioma increased succinate signaling through SUCNR1. development. Whether suppression of PNMT, KRT19,or other genes by CpG island or histone methylation Inhibition of neuronal apoptosis linked to PhD3 provides an advantage in SDH-mutated tumor progression, however, remains to be directly demonstrated. As It has been suggested that succinate accumulation previously discussed, broad overlap in hypoxia-related following SDH inactivation inhibits PHD3 activity, gene expression patterns between SDH and VHL para- which is required for neuronal apoptosis after NGF gangliomas and between genetic and sporadic HNPGL withdrawal (Lee et al. 2005). According to this model, suggests that the role of histone and DNA methylation in abnormal NGF signaling leading to reduced apoptosis and influencing global gene expression profiles may be limited. enhanced survival of sympathetic neurons provides a It is conceivable that whereas initiation of SDH-mutated unifying model for the mechanism of pheochromocytoma paragangliomas may involveconstitutivehypoxia- formation following mutations in NF1, RET, SDH, and signaling, succinate-inhibition of a-KG-dependent dioxy- VHL. Notably, Lee et al. (2005) did not observe HIF genases may contribute to tumor progression. stabilization in PC12 pheochromocytoma cell lines after Germ line mutations in FH in hereditary leiomyoma- SDH knockdown and suggested that succinate inhibition tosis and renal cell cancer, somatic gain-of-function point of PhD3, rather PhD1 which controls HIF stability,

provides a mechanistic link between SDH inactivation and cellular junction proteins (Loriot et al. 2012). Whole pheochromocytoma susceptibility (Lee et al. 2005). It is exome-sequencing identified ATRX2 mutations in subset conceivable that inhibition of neuronal apoptosis and of clinically aggressive paragangliomas, including in two epigenetic inactivation of genes important for neuronal SDHB-mutated tumors (Fishbein et al. 2015). While such differentiation by TET inactivation may collaborate to findings may help to explain the mechanism by which the promote development of paraganglioma tumors. metastatic behavior is acquired in SDHB–paragangliomas, However, it is unclear whether this common model the question remains as to why the loss of SDH through based on inhibition of apoptosis can explain the different subunit gene mutations have such distinct distinct expression profiles conferred by mutations in consequences on the metastatic potential. VHL/SDHX vs RET/NF1 in PPGLs and the activation of Because heterozygous SDH mutations predispose to hypoxia-related pathways specifically in the SDH-mutated tumor formation, haploinsufficiency of an SDH subunit paragangliomas. initiates the tumorigenic process. Hereditary paraganglioma formation usually follows loss of the unmutated SDHB or SDHD allele, which abolishes the whole mito- Other aspects of pathogenesis in chondrial complex II activity. It is conceivable that SDHB SDH-mutated paragangliomas haploinsufficiency occurs in developmentally more imma- ture paraganglionic cells that are prone to develop stem- Role of ROS in SDH-mutated pathogenesis cell like properties during tumorigenesis enabling them to Mitochondrial complex II generates significant quantities migrate and proliferate in distant sites. However, SDHD of ROS (Quinlan et al. 2012), which is further enhanced or SDHC haploinsufficiency may occur in more mature by certain mutations (Ishii et al. 2005). Whether ROS paraganglionic cells that are less likely to dedifferentiate contributes to pathogenesis of SDH-mutated paraganglio- and metastasize. mas is the subject of ongoing investigations. In addition to a et al stabilizing HIF1 (Guzy . 2008), ROS generated by Conclusion SDHC mutations has also been implicated in mutating nuclear DNA and therefore contributing to tumorigenesis Mutations in SDH subunits account for most familial and (Ishii et al. 2005). Role of somatic mutations in SDH- sporadic HNPGLs and PPGLs, and have also been linked mutated paragangliomas, however, remains unconfirmed. to other neoplasms including GISTs, renal cancer, and Endocrine-Related Cancer SDH-mutated paragangliomas does not frequently acquire pituitary adenomas. Abundant evidence suggests that point mutations in the non-mutated allele, which is often constitutive hypoxic stimulation plays an important role lost by large deletions (Dahia 2014). Recent tumor in development of SDH-mutated paraganglioma tumors. sequencing studies also show very low levels of overall However, mechanisms by which SDH regulates oxygen mutations in SDH-mutated paragangliomas (Castro-Vega sensing and signaling are poorly understood. Progress et al. 2015). would be facilitated by development of relevant animal or cell culture models that link SDH dysfunction to tumor formation and/or to altered physiological responses to Malignancy among SDHB mutation carriers hypoxia. Whether HIF1a/HIF2a is involved in the hypoxic Prevalence of malignant paragangliomas as defined by signaling pathway suspected to drive SDH-mutated metastasis among SDHB mutation carriers is substantially paragangliomas can be rigorously addressed only through higher than among SDHD carriers (13% vs 4%; van such models. Recent studies on Fh1 mouse model suggest Hulsteijn et al. 2012). The association of SDHB mutations that the stabilization of HIFa in tumor samples does not with malignancy appears to hold both for HNPGL and necessarily indicate its involvement in tumor patho- non-HNPGL (Boedeker et al. 2007). Metastasis is thought genesis. Although the association of activating EPA- to occur through a process called epithelial–mesenchymal S1/HIF2A mutations with PPGL would support a role for transition (EMT; Scheel & Weinberg 2012). EMT confers HIFs, the co-occurrence of erythrocytosis in certain cancer cells with stem-cell like properties including the carriers suggests that pathogenesis of PPGL tumors with ability to migrate and grow in distant anatomic sites. Gene EPAS1/HIF2A mutations may be more closely associated expression analyses of SDHB-related metastatic paragan- with VHL-mutated than SDHX-mutated tumors. Succinate gliomas show differential alterations in genes implicated accumulation in SDH-mutated paragangliomas inhibits in EMT, such as those encoding metalloproteinases and certain a-KG-dependent dioxygenases and leads to histone

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Received in ﬁnal form 24 June 2015 Accepted 25 June 2015 Made available online as an Accepted Preprint 25 June 2015